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目的构建人巨细胞病毒(HCMV)Han株US12基因缺失的BAC毒株,研究US12产物对HCMV Han株在人胚肺成纤维细胞(HELF)中复制的影响。方法以侧翼含有US12基因同源序列的PCR引物扩增卡那霉素抗性基因,电转至含有Han株BAC质粒的大肠杆菌DY380-Han-wt-BAC,PCR及DNA测序验证US12缺失的病毒序列。将缺失突变质粒Han-ΔUS12-BAC-P电转至HELF,获得感染性病毒颗粒Han-ΔUS12-BAC。以MOI为0.1的Han-ΔUS12-BAC毒株及Han-wt-BAC毒株感染HELF,TCID50法测定上清中病毒滴度,绘制病毒增殖曲线。结果成功构建了Han-ΔUS12-BAC克隆,转染HELF后成功获得感染性病毒。生长曲线实验结果表明,ΔUS12缺失和野生型毒株在HELF中复制增殖与扩散水平无明显差异。结论 US12为HCMV临床株Han在HELF中增殖和扩散过程中的非必需基因。
OBJECTIVE: To construct human cytomegalovirus (HCMV) Han strain with deletion of US12 gene from BAC strain and study the effect of US12 on the replication of HCMV Han strain in human embryonic lung fibroblasts (HELF). Methods The kanamycin resistance gene flanked by PCR primers containing the homologue of the US12 gene was amplified and transformed into E. coli DY380-Han-wt-BAC containing the Han strain BAC plasmid. PCR and DNA sequencing verified the deletion of the US12 deleted virus sequence . The deletion mutant plasmid Han-ΔUS12-BAC-P was electroporated to HELF to obtain the infectious virus particle Han-ΔUS12-BAC. HELF was infected with Han-ΔUS12-BAC strain and Han-wt-BAC strain with an MOI of 0.1. The virus titer in the supernatant was determined by TCID50 method and the virus proliferation curve was drawn. Results The Han-ΔUS12-BAC clone was successfully constructed and the infectious virus was successfully obtained after transfection with HELF. The results of growth curve experiment showed that there was no significant difference in the proliferation, proliferation and proliferation of ΔUS12 deletion and wild-type strain in HELF. Conclusion US12 is a non-essential gene in the process of proliferation and proliferation of HCMV Han strain in HELF.